Hydrogen peroxide production from glycerol metabolism is dispensable for virulence of Mycoplasma gallisepticum in the tracheas of chickens.

Hydrogen peroxide (H2O2) is a by-product of glycerol metabolism in mycoplasmas and has been shown to cause cytotoxicity for cocultured eukaryotic cells. There appears to be selective pressure for mycoplasmas to retain the genes needed for glycerol metabolism. This has generated interest and speculation as to their function during infection. However, the actual effects of glycerol metabolism and H2O2 production on virulence in vivo have never been assessed in any Mycoplasma species. To this end, we determined that the wild-type (WT) R(low) strain of the avian pathogen Mycoplasma gallisepticum is capable of producing H2O2 when grown in glycerol and is cytotoxic to eukaryotic cells in culture. Transposon mutants with mutations in the genes present in the glycerol transport and utilization pathway, namely, glpO, glpK, and glpF, were identified. All mutants assessed were incapable of producing H2O2 and were not cytotoxic when grown in glycerol. We also determined that vaccine strains ts-11 and 6/85 produce little to no H2O2 when grown in glycerol, while the naturally attenuated F strain does produce H2O2. Chickens were infected with one of two glpO mutants, a glpK mutant, R(low), or growth medium, and tracheal mucosal thickness and lesion scores were assessed. Interestingly, all glp mutants were reproducibly virulent in the respiratory tracts of the chickens. Thus, there appears to be no link between glycerol metabolism/H2O2 production/cytotoxicity and virulence for this Mycoplasma species in its natural host. However, it is possible that glycerol metabolism is required by M. gallisepticum in a niche that we have yet to study.